New and innovative strategies in order to overcome antibiotic resistance: a focus on phage therapy and fecal microbiota transplantation

The concept behind this approach is that, through the use of antibiotics gut microbiota might be damaged favoring the uprising of resistant pathogens; restoring microbiome diversity through the transplantation, would reduce the risk of antibiotic resistant organisms’ colonization and also the transmission to other people (Woodworth, Hayden, Young, & Kwon, 2019).
Fecal microbiota transplantation it’s becoming an accepted method for the restoration of a disrupted microbiota. A fecal suspension is prepared from a healthy donor and introduced in the gastrointestinal tract of a diseased person by oral capsule, enemas, duodenal infusion through a nasointestinal tube or through colonoscopy.
FMT enhances microbial diversity, and it has been found that treated patients’ microbiota resembles that of the healthy donor (Joint Research Centre, 2018).
A European consensus conference, held in 2016 with 28 experts from 10 countries, had the aim to provide scientifically based guidelines with the best available evidence regarding FMT indications, donor selection, preparation of fecal material, clinical management and fecal delivery and basic requirements in order to enhance an FMT center. Thanks to this consensus, the implementation of FMT centers for the treatment of C.difficile infection (CDI) is strongly recommended.
Results of the working groups: there is a high evidence that faecal microbiota infusion from a healthy donor can restore the healthy microbial flora in people with recurrent CDI leading to the resolution of symptoms. In two small randomized controlled trials (RCT), FMT showed more than 90% of resolution within respect to Vancomycin; in another larger double-blind RCT the efficacy rate was above 80% and there were compared frozen to fresh FMT, both with excellent results. In all the performed studies, FMT showed an excellent safety profile in the short-term follow up; long-term follow up are missing due to lack of reporting.
The evidence of successful FMT for recurrent CDI, creates strong basis for recommending FMT also as a treatment option for refractory CDI which might develop to be a life-threatening disease, but further studies will be needed in order to confirm this (Cammarota, et al., 2017).

Preparation of faecal material:
The preparation should be standardized but actually there are several burdens that do not allow that, among which the incomplete characterization of the material delivered into the patient or the unknown effect that oxygen exposure may trigger or even the risk of transferring microbial pathogens or undesired disease phenotypes such as obesity, metabolic syndrome and fatty liver (Antushevich, 2020).
There are no reported studies that may compare different preparation protocols of fresh material but there are statements that can be done according to the nature of microbiota itself:
• Anaerobic processing would be rationally necessary in order to treat disorders associated with microbiota alteration such as intestinal bowel
syndrome or disease because they are associated with low number of anaerobic bacteria.
• The mean amount of material used is 50 g but the weight is not a sufficient criterion in order to measure the microbiota.
• It is recommended fecal material to be diluted with sterile saline solution (0.9%) with three to five times larger volume of solvent.
• After homogenization, the solids should be drained with a gauze and the suspension should be poured into a sterile container (Cammarota, et al., 2017).

An example of preparation of fecal microbiota transplantation is described by Perez et al. in order to treat a patient with Clostridium difficile infection. The steps of the procedures are as follow:
✓ 200-300ml of diluent such as water, saline or milk added to 50-60 g of stool homogenization into a liquid suspension;
✓ Sit for 5 minutes;
✓ Filtration through the gauze and then through the syringe with filter which can be immediately used or refrigerated at 2-8° C for up to 24h or even frozen at -20° C for up to 30 days.

Donor feces can be frozen without loss of efficiency and the fecal material obtained from the donor, can be frozen at -80° C adding glycerol (Antushevich, 2020). The storage should be at -80°C because it has been observed an activation of enzymes that might lead to the degradation of sensitive microbial populations (Cammarota, et al., 2017).
Frozen faces are essential for the development of a stool bank in order to have the availability of stool on demand without the needing of screening again. As for the fresh preparations, anaerobic preparations might be more relevant for the treatment of infections other than recurrent CDI. It is recommended to add glycerol up to a final concentration of 10% because it can protect microbial cells from the damage induced by freezing unlike ethanol which instead kills the pathogens and can alter the composition of commensal microbiota such as eliminating bacteriophages, fungi and non-sporulating bacterial components (Cammarota, et al., 2017).
Stool banks have been established in some countries such as OpenBiome and AdvancingBio in the United States, Taymount Clinic in the United Kingdom, Netherlands Donor Feces Bank and the Chinese FMT bank (Joint Research Centre, 2018).
On the day of the administration, it has to be warmed up to 37° C and saline solution may be added in order to obtain the desired volume and it should be infused within 6 hours. It should be avoided repetitive freezing and warming due to the sensitivity of the microbial cells (Cammarota, et al., 2017).
The administration could be oral, nasal and rectal through colonoscopy or enema although the best way of the transplantation is considered the rectal administration through colonoscopy because the fecal substrate is deep in the cecum and there is no risk of quick removal (that happens instead with the enema).
Introduction of the fecal microbiota in the upperparts of the digestive and respiratory tracts, causes pulmonary or gastrointestinal complications such as abdominal discomfort, cramping, constipation, flatulence, bloating, belching, abdominal swelling, nausea, vomiting, diarrhea, blood in stool, dysbiosis, temperature reading. These side effects are just the normal response of the body to the introduction of live microorganisms, but they quickly disappear without triggering an invalidating condition to the patient (Antushevich, 2020).
In order to receive the faecal material, the patient’s intestine should be prepared. Patients with recurrent CDI, should be treated with vancomycin or fidaxomicin at least for 3 days before the transplant in order to repress the overabundance of C.difficile and the antibiotics should be stopped 12-48 hours before the infusion (Cammarota, et al., 2017).

Donor Selection:
The objective of the selection of a healthy donor is to avoid and prevent any adverse event related to the infused faecal material. The first selecting criteria are those based on those of the European Commission for the selection of allogenic living donors of human
tissue transplants (figure 1); furthermore, the donors undergo blood and stool testing 4 weeks before donation that include CMV, EBV, HAV, HBV, HCV, HEV, Syphilis, HIV-1 and HIV-2, Entamoeba histolytica, complete blood cell count with differential, C-RP and Erythrocyte sedimentation rate, Albumin, Creatinine and electrolytes, Aminotransferases, bilirubin, gamma-glutamyltransferase, alkaline phosphatase.
Feces are tested for detection of pathogens such as C.difficile, Salmonella, Shigella, Campylobacter, E. coli 0157 H7, Yersinia, VRE, MRSA, gram-negative multidrug- resistant bacteria, Norovirus, Giardia Lamblia, Criptosporidium parvum, Protozoa and Helminths, faecal occult blood testing (Cammarota, et al., 2017) (EUR-Lex).
An inappropriate donor screening and the incorrect analysis of the fecal donor material may lead to contamination of the patient by pathogenic microorganisms up to the development of serious adverse effects, namely induction of chronic disease in recipients. i.e., if the donor suffers from obesity, Parkinson and cancer, these diseases could be induced in the patient.
Hence, side effects of FMT, might be caused by the inaccurate analysis of fecal donor material with exacerbation of chronic disease in treated patient (Antushevich, 2020).
In 2013 the Food and Drug Administration (FDA) and the National Institutes of Health (NIH) held a joint workshop regarding the use of fecal microbiota for transplantation which was approved for treating infections of C.difficile which do not respond to standard therapies. In June 2019, the FDA issued a safety alert about the potential risks of serious and life-threatening infections due to the wrong selection of the donors: two immunocompromised patients received investigational FMT and developed invasive infections of antibiotic resistant E. coli, one of them died. Hence, the FDA determined that further precautions are needed in the selection of the healthy donor such as the screening for multi-drug-resistant organism (MDRO) risk and carriage. The procedure excludes the individuals with increased risk of exposure to resistant organisms like health care workers and recently hospitalized people; the organisms tested are: ESBL-producing Enterobacteriaceae, Vancomycin-resistant Enterococci (VRE), carbapenem-resistant Enterobacteriaceae (CRE) and Methicillin- resistant Staphylococcus Aureus (MRSA) (Carlson, 2020).